DESC:SUPERPARAMAGNETIC NANOPARTICLES MEDIATED HEMATOLOGICAL ANALYSIS OF CELLS
FIELD OF INVENTION
The invention generally relates to the field of analytical and quantitative cytology and particularly to a method for rapid and cost-effective analysis of hematological cells.
BACKGROUND
Analysis of cells includes but is not limited to physical and biological characterization. Physical characterization includes but is not limited to estimation of size in terms of dimensions and mass, and counting the number of cells in a given volume of liquid containing the cells. Various methods are known to count the number of cells. Examples of counting include manual counting, automated counting and indirect cell counting. Counting of cells is critical, specifically in case of a life endangering disease. Specifically, the cell counting becomes critical in the viral induced diseases. Examples of viral induced disease includes but is not limited to HIV induced AIDS, Dengue, Ebola and Hepatitis. It is of utmost importance to have an accurate count of the number of cells, subsequent a treatment for a viral disease. The count of the cells and the ratios of different cell types is an indication of effectiveness of the treatment.
Various counting methods are known in the art for counting the cells, subsequent to both infection and the treatment following the infection. Clinically, the method adopted for counting the number of cells and its subtypes is flow cytometry. Flow cytometry provides an accurate differential cell count. Flow cytometry works on the principle of fluorescently-labelled antibodies and counting the cells in flow. Flow cytometers are bulky, expensive, technically demanding, have a high maintenance cost, and the cost per test is also high. There are single-platform flow cytometers which are comparatively cheaper than the regular flow cytometers, but the limitation of these systems, is that they can only be used to count CD4+ and CD8+ T-cells.
There are other methods to count CD4+ T-cells such as cell sorting. Cell sorting is an effective method to separate target CD4+ T-cells from a whole blood sample. There are two dominant methods of cell sorting, namely, Fluorescence-activated cell sorting, FACS and Magnetic-activated cell sorting, MACS. In MACS, magnetic microbeads are conjugated with antibodies that are specific to a target cell. The conjugation of antibodies to beads is achieved by activating the surface functional groups. The conjugated antibody-bead complex adheres to the target cells' surface receptors. A suitable magnet is then used to separate the magnetic beads conjugated target cells from the sample. The strength of the magnetic field for cell separation depends on the size of magnetic beads conjugated to the target cells. The steps involved to separate target cells from a mixed population are complicated, since the larger density beads may interfere with binding to the cells. Additionally, the reaction will be slower between the magnetic bead and larger particles and require more incubation time, larger magnetic beads can also interfere with optical properties of the cell. These beads are expensive and laborious and limit its usage in resource-limited settings and are technically demanding. Hence, there is a need for a method that requires less preparation time and has ease of estimation.
BRIEF DESCRIPTION OF DRAWINGS
So that the manner in which the recited features of the invention can be understood in detail, some of the embodiments are illustrated in the appended drawings. It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, for the invention may admit to other equally effective embodiments.
FIG.1 shows a flowchart of the method for analysis of cells, according to an embodiment of the invention.
FIG.2 shows a graph of number of CD4+ T-cells counted from different constituents of blood, according to an embodiment of the invention.
FIG.3 shows a volumetric optimization of a sample for obtaining maximum count of CD4+ T-cells, according to an embodiment of the invention.
FIG.4 shows incubation time dependent count of CD4+ T-cells, according to an embodiment of the invention.
SUMMARY OF THE INVENTION
One aspect of the invention provides method for superparamagnetic iron oxide nanoparticles mediated hematological analysis of a blood sample. The method includes conjugating the superparamagnetic iron oxide nanoparticles with antibodies specific to a given target cell, incubating the antibody conjugated superparamagnetic iron oxide nanoparticles with the blood sample containing the target cells. Subsequent to incubation, a definite volume of the incubated blood samples containing target cells present in the blood sample is segregated through application of a magnetic field. Post segregation, the blood samples are analyzed for segregated and unsegregated volumes of the blood samples to specifically identify the target cells.
DETAILED DESCRIPTION OF THE INVENTION
Various embodiments of the invention provide a method for superparamagnetic iron oxide nanoparticles mediated haematological analysis of cells. The method includes conjugating the superparamagnetic iron oxide nanoparticles with antibodies specific to a given target cell, incubating the antibody conjugated superparamagnetic iron oxide nanoparticles with the blood sample containing the target cells. Subsequent to incubation, a definite volume of the incubated blood samples containing target cells present in the blood sample is segregated through application of a magnetic field. Post segregation, the blood samples are analyzed for segregated and unsegregated volumes of the blood samples to specifically identify the target cells. In one embodiment of the invention, the differential count obtained between the analysis of the segregated volume and the unsegregated volume yields the final count of the target cells.
FIG.1 shows a flowchart of the method for quantitative analysis of cells, according to an embodiment of the invention. Initially, 101 includes formation of superparamagnetic iron oxide nanoparticles, hereinafter referred to as SPIONs and conjugation of the formed nanoparticles with antibodies specific to a given target cells. Target cells includes but is not limited toCD4+ T-cell, CD3, CD8, CD10.103 is incubation of the antibody conjugated SPIONs with a blood sample containing the target cells. In one example of the invention, the target cells are CD4+ T-cells. The incubation of the antibody conjugated superparamagnetic iron oxide nanoparticles with the blood sample containing the target cells is carried out for a time period of 15 minutes to 60 minutes. Subsequent to incubation, the sample is separated into two vials, with one treated as control and the other taken to the next step of segregation. 105 is segregation of CD4+ T-cells inside the sample through application of a magnetic field. The vial marked for segregation is subjected to magnetic field and the supernatant fluid, post segregation is taken for counting. 107 is the counting of the supernatant fluid and the control. The control vial with unsegregated volume and the vial containing the supernatant fluid are then independently counted in an automated hematology analyser. The analyser provides a count of the cells in both the vials, a difference in count of which yields the number of target cells, present in a given sample. Each of these steps, briefly described herein above shall be explained in detail.

Synthesis and Functionalization of SPIONs:
Iron Oxide Nanoparticles are synthesized using coprecipitation technique. Coprecipitation involves reaction between Iron(III) chloride hexahydrate, Iron(II) Chloride Tetrahydrate with ammonia solution, NH4OH at a temperature in the range of about 20º C to about 40º C and air atmospheric conditions in the range of about 90 kPa to about 180 kPa. In one example of the invention, the reaction is carried out at a temperature of 25º C and a pressure of 101.3 KPa. Molar ratio of Fe2+ : Fe3+ plays important role in tuning the composition, particle size and magnetization of the material. Molar ratio of Fe2+ : Fe3+ used in the invention is in the range of about 1.2, 1.1, 2.1 and 3.1. In one example of the invention 1:3 molar ratio of Fe2+ : Fe3+ is taken.1M (range 0.5M to 1.5M) Ammonia solution is added dropwise to a mixture of Iron salts in aqueous medium. The reaction is continued under mechanical stirring for 30 mins and change in solution color during reaction indicated the formation of the particles. A Neodymium magnet with field strength of 0.5 T is used for decantation process and the precipitate is washed few times with water and ethanol in the presence of magnet. The precipitate is dried in vacuum condition and further used for amine functionalization.
Typically 0.35 g of iron oxide nanoparticles are dispersed in mixture of water and ethanol in the ration 1:40 (v/v) and sonicated for 20 mins. 1 ml of ammonia solution is added to the mixture and sonicated further for 20 mins. The nanoparticles are well dispersed in the solution after sonication process and further reaction is carried in 100 mL RB flask, 1.2 mL of (3-Aminopropyl) triethoxysilane is added dropwise to the solution for duration of 15 mins and allowed to mix under mechanical stirring process for 12 hours. A magnet with high field strength is used for separating the functionalized nanoparticles from supernatant and finally 2 mL of amine functionalized iron oxide nanoparticles are obtained, this sample is dispersed in ethanol and used for bioconjugation process without any further treatment. In one example of the invention, stable maghemite nanoparticles ??-Fe2O3 nanoparticles with particle size range of 15nm to 20 nm and magnetization in the range of about 58 emu/g is used, owing to its stability even after bioconjugation for more than six months. The functionalized SPIONs are then characterized, physically, through known characterization techniques. In one embodiment of the invention, the superparamagnetic iron oxide nanoparticle has particle size range of 15 nm to 20 nm.
Subsequent to characterization, the SPIONs obtained are calibrated for the quantity to be used for counting of target cells. FIG.2 shows a graph of percentage of CD4+ T-cells counted with respect to total white blood cells from different sample processing method of blood, according to an embodiment of the invention. From the plot it infers that the efficiency of CD4+T-cells counting in isolated WBCs is higher but in the control sample the actual percentage of CD4+ T-cells in whole blood is 12.063%. The increase in percentage of CD4+ T-cells is due to enrichment of lymphocyte cells (broader category of CD4+T-cells and other cells) and loss of other cells like neutrophils. Using whole blood without any pre-processing results in poor recovery of CD4+T-cells due to interference from high concentration of cells. Predilution of whole blood results in the better counting efficiency and the percentage of CD4+ T-cells with respect to WBCs are similar as standard test result.
EXAMPLE 1: CD4+T-cell separation using SPIONs-CD4
Several identical controls with NS(Normal Saline) and respective test samples with SPIONs-CD4 are prepared. Subsequent to incubation of the samples for a period in the range of about 15 mins to about 60 mins and mixing the samples at around every 10 minutes for proper conjugation, a Neodymium magnet is used to pellet out the CD4+T-cells bounded to SPIONs-CD4 and unbound SPIONs-CD4. The number of WBCs present in these samples, before and after magnetic separation, is counted using an automated hematology analyzer. The relative difference between the number of WBCs present in the control and the respective test sample (supernatant) are calculated. A significant difference in the WBCs is observed which is comparable with the theoretical value of CD4+T-cells present in a healthy individual from the literature. FIG.3 shows a volumetric optimization of concentration of SPION-CD4 sample for obtaining maximum count of CD4+ T-cells, according to an embodiment of the invention.
The volume of SPIONs-CD4 solution is added to prediluted whole blood at different concentrations and CD4+T-cells count is being monitored. The peak efficiency is obtained at 75µL/mL of SPIONs-CD4 concentration as observed from the graph (FIG 3). The optimum range of SPIONs-CD4 concentration is in the range between 25-50µL/mL. As the CD4+T-cell count with respect to this range is close to the data obtained from the standard analytical methods.
FIG.4 shows time dependent count of CD4+ T-cells, according to an embodiment of the invention.
Incubation time optimization data is presented in FIG4. The minimum time required for SPIONs-CD4 complex to bind to CD4+T-cells is 30 minutes. Incubating the complex and diluted blood from longer period of time doesn’t change the count of CD4+T-cells significantly. This reduction in incubation time results in short turn-around-time and the total assay can be completed in less time.
The foregoing description of the invention has been set merely to illustrate the invention and is not intended to be limiting. Since modifications of the disclosed embodiments incorporating the spirit and substance of the invention may occur to person skilled in the art, the invention should be construed to include everything within the scope of the appended claims and equivalents thereof.
,CLAIMS:WE CLAIM:
1. A method for superparamagnetic nanoparticles mediated hematological analysis of a blood sample, the method comprising of:
a. conjugating the superparamagnetic iron oxide nanoparticles with antibodies specific to a given target cell;
b. incubating the antibody conjugated superparamagnetic iron oxide nanoparticles with the blood sample containing the target cells;
c. segregating a definite volume of the incubated blood samples containing target cells present in the blood sample through application of a magnetic field; and
d. Analyzing the segregated and unsegregated volumes of the blood samples to specifically identify the target cells,
wherein the differential count obtained between the analysis of the segregated volume and the unsegregated volume yields the final count of the target cells.
2. The method of claim 1, wherein the target cell is a CD4+ T-cell, CD3, CD8,CD10.
3. The method of claim 1, wherein the antibody is specific to the selected target cell.
4. The method of claim 1, wherein the superparamagnetic iron oxide nanoparticles has particle size range of 15 nm to 20 nm.